Recombinant lubricin molecules and uses thereof

ABSTRACT

Recombinant lubricin molecules and uses thereof. Novel recombinant lubricin molecules and their uses as lubricants, anti-adhesive agents and/or intra-articular supplements for, e.g., synovial joints, meniscus, tendon, peritoneum, pericardium and pleura, are provided.

The invention relates to novel recombinant lubricin molecules and theiruses as lubricants, anti-adhesive agents and/or intra-articularsupplements for, e.g., synovial joints, meniscus, tendon, peritoneum,pericardium and pleura.

BACKGROUND OF THE INVENTION

Optimal functionality of synovial joints is dependent upon extremely lowcoefficients of friction between articulating tissues. Normally, acontiguous, well-lubricated surface is maintained on articularcartilage. During osteoarthritis (OA), however, reduced lubricationcontributes to cartilage matrix degradation and fibrillation; these inturn contribute to joint dysfunction and pain. Reduced lubrication alsoleads to joint dysfunction and pain in other forms of arthritis,including rheumatoid arthritis (RA).

For other tissues (e.g., tendons), a lubricated surface also contributesto optimal functionality. In addition to requiring a lubricated surface,normal tendon function requires the prevention of cellular adhesion totendon surfaces. In flexor tendon injury and repair, for example, theformation of tendon adhesions is the most common complication.

Native lubricin protein is related to megakaryocyte stimulating factor(MSF) precursor protein. PRG4 (proteoglycan 4) is the name for MSF thathas been accepted for the UCL/HGNC/HUGO Human Gene Nomenclaturedatabase. PRG4 protein (i.e., the MSF precursor protein) is described inU.S. Pat. No. 6,433,142 and US20020137894 (all patents and patentapplications cited in this document are incorporated by reference intheir entirety). Polypeptide encoded by exon 6 of the PRG4 gene isheavily glycosylated and appears necessary for a PRG4-related protein toserve as a lubricant, e.g., between surfaces of articular cartilage.

Studies indicate that PRG4 glycoprotein is also synthesized by theintimal synoviocytes that line tendon sheaths; it is highly likely thatthe glycoprotein also originates from tenocytes (Rees et al., 2002). Theglycoprotein is prominently present in fibrocartilaginous regions oftendon. In a manner complementary to its synovial-fluid function, theglycoprotein may play an important cytoprotective role for tendons bypreventing cellular adhesion to tendon surfaces, as well as by providinglubrication during normal tendon function.

Exon 6 of the PRG4 (also called “lubricin”) gene encodes approximately76-78 repeats of KEPAPTT-similar sequences and 6 repeats of XXTTTX-likesequences. Varying the number of comparable repeat sequences inrecombinant lubricin proteins according to the present invention allowsfor development of improved biotherapeutics for enhancing lubrication injoints and for countering undesired adhesion between tissues.

SUMMARY OF THE INVENTION

The present invention relates to novel recombinant lubricin moleculesand their use as lubricants, anti-adhesive agents and/or intra-articularsupplements.

In order to optimize expression parameters and investigate thefunctional necessity of all approximately 76-78 KEPAPTT-similarsequences, lubricin expression constructs were designed which enabledthe synthesis of recombinant lubricin proteins with varying degrees ofO-linked oligosaccharide substitution. This is accomplished byincorporating variable numbers of the KEPAPTT-like sequences into a“core” cDNA construct comprised of exons 1 through 5, 5′- and3′-flanking regions of exon 6, and exons 7 through 12. Iterativeinsertion of “synthetic cDNA cassettes” encoding multiple KEPAPTT-likesequences facilitates the generation of recombinant lubricin constructsof different sizes. The initial focus of these studies was on constructPRG4-Lub:1 (containing DNA of “synthetic cDNA cassette-1” (SEQ ID NO:1), which encodes four KEPAPTT sequences).

The recombinant lubricin proteins of the present invention share primarystructure with several isoforms of native human lubricin (see U.S. Pat.No. 6,743,774, US20040072741, and WO0064930). Among characterizedisoforms, each isoform differs in the composition of PRG4 gene exonsthat encode the isoform's primary structure. For example, exons 1through 12 of the PRG4 gene encode the V0 isoform, which represents thefull-length isoform, while exons 1 through 4 and 6 through 12 encode theV1 isoform, which lacks only a segment encoded by exon 5. Exons 1through 3 and 6 through 12 encode the V2 isoform, which lacks segmentsencoded by exons 4 and 5. Finally, exons 1, 3, and 6 through 12 encodethe V3 isoform, which lacks segments encoded by exons 2, 4, and 5. Otherisoforms likely exist, and some related mutant proteins have beendescribed (see US20020086824).

In particular, the present invention provides recombinant lubricinprotein comprising repetitive KEPAPTT-like sequences. In preferredembodiments, the invention provides isolated protein comprising SEQ IDNOS: 9, 13, 17, 21 or 25. The invention provides in related embodimentsisolated protein comprising SEQ ID NOS: 7, 11, 15, 19 or 23. In furtherrelated embodiments, the invention provides isolated polynucleotidecomprising nucleic acid sequence encoding recombinant lubricin protein.In preferred embodiments, the invention provides isolated polynucleotidecomprising nucleic acid sequence encoding the protein. In furtherrelated embodiments, the invention provides isolated polynucleotidehaving at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ IDNOS: 6, 10, 14, 18 or 22 over the entire length of the sequence.

In related aspects, the present invention also provides an isolatedprotein comprising SEQ ID NO: 26 joined to (N minus 2) repeat(s) of SEQID NO: 27, where N equals an integer from 3 through 200. In furtherrelated embodiments, the present invention provides an isolated proteincomprising SEQ ID NO: 26 plus SEQ ID NO: 28 plus [(N minus 2) repeat(s)of SEQ ID NO: 27] plus SEQ ID NO: 29, where N equals an integer from 3through 200. In embodiments of the related aspects of the inventionnoted in this paragraph, more preferably N equals an integer from 5through 50, and even more preferably N equals an integer from 10 through30.

TABLE 1 Identification of Sequences Having Sequence Identifiers SEQ IDNO: Identification 1 nucleotide sequence of synthetic cDNA cassette-1:155 bases 2 translation of SEQ ID NO: 1: 51 amino acids 3 nucleotidesequence of synthetic cDNA cassette-2: 125 bases 4 translation of SEQ IDNO: 3: 41 amino acids 5 pTmed2 vector containing recombinant PRG4-Lub: 1cDNA construct: 8049 bases 6 recombinant PRG4-Lub: 1 cDNA construct:2946 bases 7 amino acid sequence of entire PRG4-LUB: 1 protein: 981amino acids 8 Lub: 1 DNA insert from synthetic cDNA cassette-1: 157bases 9 51 amino acids encoded by Lub: 1 DNA insert (4 KEPAPTT sequencesbetween S373 to E425 in SEQ ID NO: 7) 10 recombinant PRG4-Lub: 2 cDNAconstruct: 3024 bases 11 amino acid sequence of entire PRG4-LUB: 2protein: 1007 amino acids 12 Lub: 2 DNA insert from synthetic cDNAcassette-1 and one synthetic cDNA cassette-2 sequence: 235 bases 13 77amino acids encoded by Lub: 2 DNA insert (6 KEPAPTT sequences betweenS373 and E451 in SEQ ID NO: 11) 14 recombinant PRG4-Lub: 3 cDNAconstruct: 3117 bases 15 amino acid sequence of entire PRG4-LUB: 3protein: 1038 amino acids 16 Lub: 3 DNA insert from synthetic cDNAcassette-1 and two synthetic cDNA cassette-2 sequences: 328 bases 17 108amino acids encoded by Lub: 3 DNA insert (9 KEPAPTT sequences betweenS373 and E482 in SEQ ID NO: 15) 18 recombinant PRG4-Lub: 4 cDNAconstruct: 3210 bases 19 amino acid sequence of entire PRG4-LUB: 4protein: 1069 amino acids 20 Lub: 4 DNA insert from cDNA cassette-1 andthree synthetic cDNA cassette-2 sequences: 421 bases 21 139 amino acidsencoded by Lub: 4 DNA insert (12 KEPAPTT sequences between S373 and E513in SEQ ID NO: 19) 22 recombinant PRG4-Lub: 5 cDNA construct: 3303 bases23 amino acid sequence of entire PRG4-LUB: 5 protein: 1100 amino acids24 Lub: 5 DNA insert from cDNA cassette-1 and four synthetic cDNAcassette-2 sequences: 514 bases 25 170 amino acids encoded by Lub: 5 DNAinsert (15 KEPAPTT sequences between S373 and E544 in SEQ ID NO: 23) 26amino acid sequence “APTTPKEPAPTTTKSAPTTPKEPAPTTT KEPAPTTPKEPAPTTTK” (45amino acids) in preferred PRG4- LUB: N protein 27 amino acid sequence“KEPAPTTTKEPAPTTTKSAPTTP KEPAPTTP” (31 amino acids) repeated N-1 timesin preferred PRG4-LUB: N protein 28 amino acid sequence“EPAPTTTKSAPTTPKEPAPTTP” (22 amino acids) joining SEQ ID NO: 26 to (N-2)repeats of SEQ ID NO: 27 in preferred PRG4-LUB: N protein where N ≧ 3.29 amino acid sequence “KEPKPAPTTP” (10 amino acids) in preferredPRG4-LUB: N protein where N ≧ 2.

The invention also provides in related embodiments a compositioncomprising a therapeutically effective amount of a recombinant lubricinprotein in a pharmaceutically acceptable carrier. In some embodiments,the composition additionally comprises hyaluronan or hylan.

The invention further provides a method of treating a subjectcomprising: obtaining a recombinant lubricin protein composition; andadministering said composition to a tissue of the subject. In relatedembodiments of this method of the invention, the tissue is selected fromthe group consisting of cartilage, synovium, meniscus, tendon,peritoneum, pericardium, and pleura. In further related embodiments ofthis method of the invention, the method additionally comprises a stepselected from the group consisting of: providing an anesthetic to thesubject; providing an anti-inflammatory drug to the subject; providingan antibiotic to the subject; aspirating fluid from the subject; washingtissue of the subject; and imaging tissue of the subject. In otherrelated embodiments, the subject is selected from the group consistingof a mouse, a rat, a cat, a dog, a horse, and a human.

In other embodiments, the invention also provides an expression vectorcomprising a polynucleotide encoding a recombinant lubricin proteinwherein the polynucleotide is operably linked to an expression controlsequence. In related embodiments, the invention provides a method ofproducing recombinant lubricin protein comprising: growing cellstransformed with the expression vector in liquid culture media; andcollecting recombinant lubricin protein from the media. The collectingprotein step may further comprise: concentrating the protein byfiltering the media through a membrane; collecting the retained proteinfrom the membrane; and solubilizing the collected protein in a bufferedsalt solution containing L-arginine hydrochloride ranging inconcentration from 0.1 to 2.0 M.

In another related embodiment, the invention provides isolated antibodyspecific for a recombinant lubricin protein.

Other features and advantages of the invention will be apparent from thefollowing description of preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION OF THE INVENTION

The base DNA construct utilized in generating recombinant lubricinproteins may include variable arrangements of sequences 5′ and 3′ ofexon 6 of the PRG4 gene. For example, the base DNA construct may includevariable arrangements of sequences encoding somatomedin B-like domains(exons 2 through 4) or hemopexin-like domains (exons 7 through 9).

Embodiments of the base DNA construct having various exon arrangements3′ of exon 6 may include base DNA constructs that include only exon 7,8, 9, 10, 11, or 12 individually, or exon pairs (7 and 8), (7 and 9), (7and 10), (7 and 11), (7 and 12), (8 and 9), (8 and 10), (8 and 11), (8and 12), (9 and 10), (9 and 11), (9 and 12), (10 and 11), (10 and 12),or (11 and 12), or exon triplets (7, 8 and 9), (7, 8 and 10), (7, 8, and11), (7, 8, and 12), (7, 9 and 10), (7, 9 and 11), (7, 9 and 12), (7, 10and 11), (7, 10 and 12), (7, 11 and 12), (8, 9 and 10), (8, 9 and 11),(8, 9 and 12), (8, 10 and 11), (8, 10 and 12), (8, 11 and 12), (9, 10and 11), (9, 10 and 12), (9, 11 and 12), or (10, 11 and 12), or exonquadruplets (7, 8, 9 and 10), (7, 8, 9 and 11), (7, 8, 9 and 12), (7, 8,10 and 11), (7, 8, 10 and 12), (7, 8, 11 and 12), (7, 9, 10 and 11), (7,9, 10 and 12), (7, 9, 11 and 12), (7, 10, 11 and 12), (8, 9, 10 and 11),(8, 9, 10 and 12), (8, 9, 11 and 12), (8, 10, 11 and 12), or (9, 10, 11and 12), or exon quintets (7, 8, 9, 10 and 11), (7, 8, 9, 10 and 12),(7, 8, 9, 11 and 12), (7, 8, 10, 11 and 12), (7, 9, 10, 11 and 12), or(8, 9, 10, 11 and 12), or exon sextet (7, 8, 9, 10, 11 and 12).

In addition, embodiments of the base DNA construct having various exonarrangements 5′ of exon 6 may include base DNA constructs that includeonly exon 1, 2, 3, 4, or 5 individually, or exon pairs (1 and 2), (1 and3), (1 and 4), (1 and 5), (2 and 3), (2 and 4), (2 and 5), (3 and 4), (3and 5), or (4 and 5), or exon triplets (1, 2 and 3), (1, 2 and 4), (1, 2and 5), (1, 3 and 4), (1, 3 and 5), (1, 4 and 5), (2, 3 and 4), (2, 3and 5), (2, 4 and 5), or (3, 4 and 5), or exon quadruplets (1, 2, 3 and4), (1, 2, 3 and 5), (1, 2, 4 and 5), (1, 3, 4 and 5), or (2, 3, 4 and5), or exon quintets (1, 2, 3, 4 and 5).

The present invention also encompasses proteins encoded by base DNAconstructs, i.e., wherein part or all of exon 6 sequence-encodedpolypeptide is deleted and no amino acids encoded by inserts fromsynthetic cDNA cassettes have been added.

The present invention also encompasses polynucleotides that arehomologous to the specific embodiments outlined herein, e.g., having atleast 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to thespecified DNA sequences. The invention further includes polynucleotideshaving nucleic acid sequence capable of hybridizing over the length of afunctional domain to the complement of the specified DNA sequences underhigh stringency conditions. The invention also includes proteins encodedby these homologous or hybridizing polynucleotides.

In order to delineate more clearly embodiments of the present invention,the following definitions are provided.

Definitions. The phrase “repetitive KEPAPTT-like sequence” means anamino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99% orhigher identity to: (a) sequence“APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTK” (SEQ ID NO: 26; 45 aminoacids) and having at least one O-linked substitution; (b) sequence“KEPAPTTTKEPAPTTTKSAPTTPKEPAPTTP” (SEQ ID NO: 27; 31 amino acids) andhaving at least one O-linked substitution; or (c) sequence“EPAPTTTKSAPTTPKEPAPTTP” (SEQ ID NO: 28; 22 amino acids) and having atleast one O-linked substitution. A repetitive KEPAPTT-like sequence maypreferably have two, three, four or more O-linked substitutions.

While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans and has a definite meaning with respect to agiven specified method. Sequence identity described herein is measuredusing the BLAST 2 SEQUENCES tool available through NCBI(http://www.ncbi.nlm.nih.gov/blast/; see also Tatusova and Madden(1999)). For amino acid sequences, the parameters used are expect=1000;word size=2; filter=off; and other parameters set to default values.These same parameters are used for nucleic acid sequences, except wordsize=8. Default values for amino acid sequence comparisons are:Matrix=BLOSUM62; open gap=11; extension gap=1 penalties; andgap×dropoff=50. Default values for nucleic acid sequence comparisonsare: reward for a match=1; penalty for a mismatch=−2; strand option=bothstrands; open gap=5; extension gap=2 penalties; and gap×dropoff=50.

An O-linked substitution of recombinant lubricin may be a substitutionwith the lubricating oligosaccharide β-(1-3)-Gal-GalNac, or with othermoieties, including artificial or naturally-occurring carbohydratemoieties (such as keratan sulfate or chondroitin sulfate). In someembodiments, the O-linked substitution may be with moieties thatcontribute to a capacity of recombinant lubricin to act as a carrier ofsurface active phospholipid (SAPL) or surfactants (Hills, 2002). Percentglycosylation or substitution is determined by weight (dry weight).

High stringency conditions, when used in reference to DNA:DNAhybridization, comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5× SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1× SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

Polypeptides or other compounds described herein are said to be“isolated” when they are within preparations that are at least 50% byweight (dry weight) the compound of interest. Polypeptides or othercompounds described herein are said to be “substantially pure” when theyare within preparations that are at least 80% by weight (dry weight) thecompound of interest. Polypeptides or other compounds described hereinare said to be “homogeneous” when they are within preparations that areat least 95%, and preferably 99%, by weight (dry weight) the compound ofinterest. Purity is measured by reducing polyacrylamide gelelectrophoresis and enhanced coomassie blue staining, followed byoptical density traces of bands (i.e., with protein purity beingmeasured through optical densitometry).

“Pyrogen-free” means free of fever causing contaminants, includingendotoxin. Measurement of contaminants is to be performed by theapplicable standard tests set by the U.S. Food and Drug Administration.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the relevant pharmaceuticalcomposition or method that is sufficient to show a meaningful patientbenefit, i.e., treatment, healing, prevention or amelioration of therelevant medical condition, or an increase in rate of treatment,healing, prevention or amelioration of such conditions. When applied toan individual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

Embodiments of the present invention may be used as intra-articularsupplements. Intra-articular supplementation with compounds not derivedfrom lubricin has been practiced as a joint therapy. For example,“viscosupplementation” with polymeric hyaluronan (HA) and highermolecular weight hylans (such as SYNVISC® elastoviscous fluid “Hylan G-F20”-distributed by WYETH® Pharmaceuticals) is used clinically to treatOA-associated knee pain. This viscosupplementation has shown significanttherapeutic value, particularly in reducing weight-bearing pain inpatients (Wobig et al., 1998).

Hylan G-F 20 is generated by cross-linking several HA molecules obtainedfrom rooster or chicken combs. Viscosupplementation with Hylan G-F 20can be significantly more efficacious for alleviating pain thanviscosupplementation with lower molecular weight HA (Wobig et al.,1999). In addition, relieving pain by viscosupplementation with HylanG-F 20 may be particularly preferable to administration of NSAIDs forthose patients who do not tolerate NSAIDs (e.g., in patients with a highrisk of gastrointestinal complications; Espallargues and Pons, 2003).Though Hylan G-F 20 viscosupplementation is a safe and well-toleratedtherapy that provides a short-term (i.e., until 3-6 monthsposttreatment) decrease in pair symptoms while improving joint function,the therapy may not significantly forestall the eventual need for kneereplacement in OA patients (Espallargues and Pons, 2003).

EXAMPLE 1 Cloning of Recombinant Lubricin

Constructs. In some embodiments, the base DNA construct for thegeneration of recombinant lubricin molecules is composed of the Metcodon (ATG) through the BssHII restriction site (ĜCGCGC) of SEQ ID NO: 6(i.e., base nos. 1 through 1123) and the BspEI restriction site (T̂CCGGA)through the stop codon (TAA) of SEQ ID NO: 6 (i.e., base nos. 1269through 2946). These sequences, i.e., base nos. 1 through 1123 and 1269through 2946 of SEQ ID NO: 6, encode amino acids M1 through 5373(encoded by exons 1 through 5 and approximately 174 flanking 5′-codonsof exon 6) and E848 through P1404 (encoded by approximately 293 flanking3′-codons of exon 6 and exons 7 through 14) of native full-lengthlubricin (i.e., PRG4). The portion of exon 6 absent from the base DNAconstruct corresponds to DNA sequence encoding amino acids A374 throughP847 of native PRG4 (474 amino acids absent out of approximately 940amino acids encoded by exon 6). This absent amino acid sequence is richin KEPAPTT-like sequences.

DNA sequence of synthetic cDNA cassette-1 (SEQ ID NO: 1) is addedBssHII/BspEI to the base construct to make the recombinant PRG4-Lub:1cDNA construct (SEQ ID NO: 6). SEQ ID NO: 6 is composed of the Lub:1 DNAinsert (SEQ ID NO: 8; which encodes the 51 amino acids of SEQ ID NO: 9with its four KEPAPTT sequences) between DNA encoding amino acids M1through 5373 and DNA encoding E848 through P1404 of native PRG4. Inother words, in place of A374 through P847 (474 amino acids) of nativePRG4, the recombinant lubricin PRG4-LUB:1 includes 51 amino acids thatform four perfect KEPAPTT sequences and approximately three imperfectKEPAPTT sequences.

DNA sequence of synthetic cDNA cassette-2 (SEQ ID NO: 3) is addedBsu36I/BspEI to the PRG4-Lub:1 construct to make the PRG4-Lub:2 cDNAconstruct (SEQ ID NO: 10). The PRG4-Lub:1 cDNA construct has one Bsu36Irestriction site (CĈTNAGG, i.e., CĈTAAGG; base nos. 1225 through 1231 ofSEQ ID NO: 6). When synthetic cDNA cassette-2 is added to the PRG4-Lub:1cDNA construct, this Bsu36I site is destroyed, but synthetic cassette-2contains another internal Bsu36I restriction site (CĈTNAGG, i.e.,CĈTAAGG; base nos. 92 through 98 of SEQ ID NO: 3). Consequently, aPRG4-Lub:N+1 construct can be made by adding synthetic cDNA cassette-2Bsu36I/BspEI to the previous PRG4-Lub:N construct at this internalBsu36I restriction site provided by synthetic cDNA cassette-2.

The cDNA cassettes are synthesized as single stranded oligonucleotidesand hybridized together to produce a double stranded DNA fragment withsticky ends. This is why the terminal BssHII, Bsu36I, and BspEI sitesappear incomplete. In synthetic cDNA cassette-1 (SEQ ID NO: 1), asequence bounded by remnant flanking BssHII (ĜCGCGC) and BspEI (T̂CCGGA)restriction sites includes an internal Bsu36I restriction site (CĈTNAGG,i.e., CĈTAAGG); the restriction sites are underlined below:

CGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAGGAGCCAGCTCCTACTACAACGAAACCGGCACCAAC CACTCCGG

SEQ ID NO: 2, which is a translation of SEQ ID NO: 1, includes fourKEPAPTT sequences that are perfect matches (highlighted below):

1   A  P  T  T  P   K  E  P  A  P  T  T   T  K  S  A  P  T  T  PCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCC 21  K  E  P  A  P  T  T   T   K  E  P  A  P  T  T   P   K  E  P  AAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAGGAGCCAGCT 41  P  T  T   T  K  P  A  P  T  T  P CCTACTACAACGAAACCGGCACCAACCACTCCGG

Synthetic cDNA cassette-2 (SEQ ID NO: 3) similarly has a remnant5′-terminal Bsu36I restriction site (i.e., CĈTNAGG, evidenced only bythe TAA sequence), a 3′-terminal remnant BspEI restriction site(T̂CCGGA), and an internal Bsu36I restriction site (CĈTNAGG); therestriction sites are underlined below:

TAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACTCCGG

SEQ ID NO: 4, which is a translation of SEQ ID NO: 3, includes threeKEPAPTT sequences that are perfect matches (highlighted below):

1    K  E  P  A  P  T  T   T   K  E  P  A  P  T  T   T  K  S  A  PTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCC 21 T  T  P   K  E  P  A  P  T  T   P  K  E  P  K  P  A  P  T  TACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACT 41  P CCGG

The recombinant PRG4-Lub:1 cDNA construct (SEQ ID NO: 6) in pTmed2vector (construct plus vector equals SEQ ID NO: 5) is flanked by SalI(ĜTCGAC; base nos. 1027 through 1032 of SEQ ID NO: 5) and NotI(GĈGGCCGC; base nos. 3984 through 3991 of SEQ ID NO: 5) restrictionsites. The SalI site incorporates a modified Kozak translationinitiation sequence (CCCACC; base nos. 1032 through 1037 of SEQ ID NO:5) before the translation start codon ATG (base nos. 1038 through 1040of SEQ ID NO: 5). Between the BssHII (ĜCGCGC; base nos. 2155 through2160 of SEQ ID NO: 5) and BspEI (T̂CCGGA; base nos. 2306 through 2311 ofSEQ ID NO: 5) restriction sites is found the internal Bsu36I cloningsite (CĈTNAGG, i.e., CĈTAAGG; base nos. 2262 through 2268 of SEQ ID NO:5).

The PRG4-Lub:1 cDNA construct (SEQ ID NO: 6) is translated into thePRG4-LUB:1 protein (SEQ ID NO: 7). The insert between 5373 and E425(i.e., E848 of native PRG4) of the entire PRG4-LUB:1 protein (SEQ ID NO:7) is the 51 amino acids of SEQ ID NO: 9. These are translated from theLub:1 DNA insert (SEQ ID NO: 8) and include four perfect KEPAPTTsequences. Between the BssHII restriction site (ĜCGCGC; base nos. 1118through 1123 of SEQ ID NO: 6) and the BspEI restriction site (T̂CCGGA;base nos. 1269 through 1274 of SEQ ID NO: 6) is found the internalBsu36I cloning site (CĈTNAGG, i.e., CĈTAAGG; base nos. 1225 through 1231of SEQ ID NO: 6).

As in the recombinant PRG4-Lub:1 construct in pTmed2 vector, therecombinant PRG4-Lub:2 cDNA construct (SEQ ID NO: 10) in pTmed2 vectoris flanked by SalI (ĜTCGAC) and NotI (GĈGGCCGC) restriction sites; theSalI site incorporates a modified Kozak translation initiation sequence(CCCACC) before the translation start codon ATG (base nos. 1 through 3of SEQ ID NO: 10). Similarly, the recombinant PRG4-Lub:3 cDNA construct(SEQ ID NO: 14), the recombinant PRG4-Lub:4 cDNA construct (SEQ ID NO:18), and the recombinant PRG4-Lub:5 cDNA construct (SEQ ID NO: 22) inpTmed2 vector are each flanked by SalI (ĜTCGAC) and NotI (GĈGGCCGC)restriction sites; the SalI site incorporates a modified Kozaktranslation initiation sequence (CCCACC) before the translation startcodon ATG (base nos. 1 through 3 of SEQ ID NOS: 14, 18, and 22,respectively).

Within the PRG4-Lub:2 cDNA construct, the internal Bsu36I cloning site(CĈTNAGG, i.e., CĈTAAGG; base nos. 1318 through 1324 of SEQ ID NO: 10)is found between the BssHII (ĜCGCGC; base nos. 1118 through 1123) andBspEI (T̂CCGGA; base nos. 1347 through 1352) restriction sites. ThePRG4-Lub:2 construct (SEQ ID NO: 10) is translated into the PRG4-LUB:2protein (SEQ ID NO: 11). The insert between S373 and E451 (i.e., E848 ofnative PRG4) of the entire PRG4-LUB:2 protein (SEQ ID NO: 11) is the 77amino acids of SEQ ID NO: 13. These are translated from the Lub:2 DNAinsert (SEQ ID NO:12). In place of A374 through P847 (474 amino acids)of native PRG4, the 77 amino acids of the recombinant lubricinPRG4-LUB:2 form six perfect KEPAPTT sequences and approximately fourimperfect KEPAPTT sequences.

Within the PRG4-Lub:3 cDNA construct, the internal Bsu36I cloning site(CĈTNAGG, i.e., CĈTAAGG; base nos. 1411 through 1417 of SEQ ID NO: 14)is found between BssHII (ĜCGCGC; base nos. 1118 through 1123) and BspEI(T̂CCGGA; base nos. 1440 through 1445) restriction sites. The PRG4-Lub:3construct (SEQ ID NO: 14) is translated into the PRG4-LUB:3 protein (SEQID NO: 15). The insert between S373 and E482 (i.e., E848 of native PRG4)of the entire PRG4-LUB:3 protein (SEQ ID NO: 15) is the 108 amino acidsof SEQ ID NO: 17. These are translated from the Lub:3 DNA insert (SEQ IDNO:16). In place of A374 through P847 (474 amino acids) of native PRG4,the 108 amino acids of the recombinant lubricin PRG4-LUB:3 form nineperfect KEPAPTT sequences and approximately five imperfect KEPAPTTsequences.

Within the PRG4-Lub:4 cDNA construct, the internal Bsu36I cloning site(CĈTNAGG, i.e., CĈTAAGG; base nos. 1504 through 1510 of SEQ ID NO: 18)is found between BssHII (ĜCGCGC; base nos. 1118 through 1123) and BspEI(T̂CCGGA; base nos. 1533 through 1538) restriction sites. The PRG4-Lub:4construct (SEQ ID NO: 18) is translated into the PRG4-LUB:4 protein (SEQID NO: 19). The insert between S373 and E513 (i.e., E848 of native PRG4)of the entire PRG4-LUB:4 protein (SEQ ID NO: 19) is the 139 amino acidsof SEQ ID NO: 21. These are translated from the Lub:4 DNA insert (SEQ IDNO:20). In place of A374 through P847 (474 amino acids) of native PRG4,the 139 amino acids of the recombinant lubricin PRG4-LUB:4 form twelveperfect KEPAPTT sequences and approximately six imperfect KEPAPTTsequences.

Within the PRG4-Lub:5 cDNA construct, the internal Bsu36I cloning site(CĈTNAGG, i.e., CĈTAAGG; base nos. 1597 through 1603 of SEQ ID NO: 22)is found between BssHII (ĜCGCGC; base nos. 1118 through 1123) and BspEI(T̂CCGGA; base nos. 1626 through 1631) restriction sites. The PRG4-Lub:5construct (SEQ ID NO: 22) is translated into the PRG4-LUB:5 protein (SEQID NO: 23). The insert between S373 and E544 (i.e., E848 of native PRG4)of the entire PRG4-LUB:5 protein (SEQ ID NO: 23) is the 170 amino acidsof SEQ ID NO: 25. These are translated from the Lub:5 DNA insert (SEQ IDNO:24). In place of A374 through P847 (474 amino acids) of native PRG4,the 170 amino acids of the recombinant lubricin PRG4-LUB:5 form fifteenperfect KEPAPTT sequences and approximately seven imperfect KEPAPTTsequences.

Importantly, the process of inserting the synthetic cDNA cassette-2. canbe iterated indefinitely. Each iteration results in the addition ofthree perfect KEPAPTT sequences. Just as recombinant lubricinsPRG4-LUB:2 through PRG4-LUB:5 are constructed in this way through theuse of insert sequences, recombinant lubricins PRG4-LUB:6 throughPRG4-LUB:N are constructed. Table 2 provides a summary of BssHII/BspEIinsert sequences.

TABLE 2 BssHII|BspE1 Insert Sequences LUB SEQ Sequences (restrictionsites underlined in DNA inserts; INSERT ID NO: KEPAPTT sequences arehighlighted in protein inserts) Lub: 1 8GCGCGCCCACAACTCCAAAAGAGGCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAGGAGCCAGCTCCTACTACAACGAAACCGGCACCAACCACTCCGGA LUB: 1 9 APTTPKEPAPTT TKSAPTTP KEPAPTT T KEPAPTT P KEPAPTT TKPAPTTP Lub: 2 12GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACTCCGGA LUB: 2 13 APTTP KEPAPTT TKSAPTTP KEPAPTT TKEPAPTT P KEPAPTT T KEPAPTT TK SAPTTP KEPAPTT PKEPKPAPTTP Lub: 3 16GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAA CCACTCCGGA LUB: 317 APTTP KEPAPTT TKSAPTTP KEPAPTT T KEPAPTT P KEPAPTT T KEPAPTT TKSAPTTP KEPAPTT P KEPAPTT T KEPAPTT TKSAPTTP KEPAPTT PKEPKPAPT TP Lub: 420 GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACTCCGGA LUB: 4 21 APTTPKEPAPTT TKSAPTTP KEPAPTT T KEPAPTT P KEPAPTT T KEPAPTT TK SAPTTP KEPAPTTP KEPAPTT T KEPAPTT TKSAPTTP KEPAPTT P KEPAPT TT KEPAPTT TKSAPTTPKEPAPTT PKEPKPAPTTP Lub: 5 24GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACTCCGGA LUB: 5 25 APTTP KEPAPTT TKSAPTTPKEPAPTT T KEPAPTT P KEPAPTT T KEPAPTT TK SAPTTP KEPAPTT P KEPAPTT TKEPAPTT TKSAPTTP KEPAPTT P KEPAPT TT KEPAPTT TKSAPTTP KEPAPTT P KEPAPTTT KEPAPTT TKSAPTTP KEPAPTT PKEPKPAPTTP

Although we have exemplified the base DNA construct with full-lengthPRG4 containing all 12 exons (minus a central portion of exon 6), splicevariants of PRG4 may also be employed, depending on the variousactivities and length desired. Additionally, different restrictionsenzymes may be employed in an analogous strategy, providing that theirlocation is conveniently located within nucleic acid sequence encodingPRG4 protein. In other embodiments, the base DNA construct lacks nativeexon 6 sequence, but includes one or more of exon 1 through exon 5sequences or of exon 7 through exon 12 sequences of the native PRG4gene. In other embodiments, the base DNA construct is identical to arecombinant MSF sequences described in U.S. Pat. No. 6,433,142 orUS20020137894 except that part or all of the sequences of exon 6 areabsent.

The invention provides cDNA constructs encoding recombinant lubricinsthat are cloned into SalI (ĜTCGAC; base nos. 1027 through 1032 of SEQ IDNO: 5) and NotI (GĈGGCCGC; base nos. 3984 through 3991 of SEQ ID NO: 5)restriction sites in the eucaryotic expression vector pTmed2 as apreferred embodiment (e.g., recombinant PRG4-Lub:1 cDNA construct inpTmed2 expression vector is located in SEQ ID NO: 5 at base nos. 1038though 3983). The SalI site incorporates the first base of a modifiedKozak translation initiation sequence (CCCACC; base no. 1032 of SEQ IDNO: 5) before the methionine start codon (ATG; base nos. 1038 through1040 of SEQ ID NO: 5). Other embodiments of the invention include otherrestriction site combinations and other expression vectors.

In a preferred embodiment, the interative process makes use of thesynthetic cDNA cassette-1 (SEQ ID NO: 1) in expression vector pTmed2,which is flanked by the restriction sites for BssHII (ĜCGCGC) and BspEI(T̂CCGGA), and the synthetic cDNA cassette-1, which includes an internalBsu36I restriction site (CĈTNAGG, i.e., CĈTAAGG; base nos. 107 to 113 ofSEQ ID NO: 1). For the iterative generation of recombinant lubricinconstructs containing KEPAPTT-like sequences in this preferredembodiment, synthetic cDNA cassette-2 (SEQ ID NO: 3) is inserted betweenthe Bsu36I and BspEI sites of the recombinant construct. Synthetic cDNAcassette-2 (SEQ ID NO: 3) is flanked by a modified remnant Bsu36I site(TAAAG) and a remnant BspEI (ACTCCGG) site. It also includes an internalBsu36I site (CĈTNAGG, i.e., CĈTAAGG; base nos. 92 through 98 of SEQ IDNO: 3). Upon cloning synthetic cDNA cassette-2 into the Bsu36I and BspEIsites of a recombinant lubricin construct, the Bsu36I cloning site ofthe original construct is destroyed leaving one unique Bsu36I cloningsite in the new construct.

In this preferred embodiment, the amino acid sequence “APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTK” (SEQ ID NO: 26; 45 amino acids)remains a part of each PRG4-LUB:N protein (where N=an integer of 1 ormore). In addition, the amino acid sequence“KEPAPTTTKEPAPTTTKSAPTTPKEPAPTTP” (SEQ ID NO: 27; 31 amino acids) isencoded by the DNA insert that becomes part of each PRG4-Lub:N+1 cDNAconstruct through the addition of synthetic cDNA cassette-2 Bsu36I/BspEIto a PRG4-Lub:N cDNA construct. For PRG4-LUB:N protein where N is aninteger greater than or equal to 3, the amino acid sequence“EPAPTTTKSAPTTPKEPAPTTP” (SEQ ID NO: 28; 22 amino acids) joins SEQ IDNO: 26 to (N minus 2) repeats of SEQ ID NO: 27 in preferred embodiments.Furthermore, the amino acid sequence “KEPKPAPTTP” (SEQ ID NO: 29; 10amino acids) immediately follows the last insert repeat of SEQ ID NO: 27in preferred embodiments of the PRG4-LUB:N protein where N is an integergreater than or equal to 2.

Because they form at least two KEPAPTT sequences, SEQ ID NO: 26, SEQ IDNO: 27, and SEQ ID NO: 28 are each designated herein to be a “repetitiveKEPAPTT-like sequence” (the N-terminus of SEQ ID 28 links to a K residueso that SEQ ID NO: 28 forms two KEPAPTT sequences in PRG4-LUB:Nproteins).

Consequently, for recombinant lubricin protein. PRG4-LUB:N (where Nequals an integer of 1 or more), the PRG4-LUB:N protein comprises SEQ IDNO: 26 in a preferred embodiment. Furthermore, for recombinant lubricinprotein PRG4-LUB:N (where N equals an integer of 2 or more), thePRG4-LUB:N protein also comprises SEQ ID NO: 27 in a preferredembodiment. SEQ ID NO: 27 is repeated (N minus 1) times within eachPRG4-LUB:N protein in these preferred embodiments. In PRG4-LUB:2, SEQ IDNO: 26 and SEQ ID NO: 27 overlap (i.e., they share a KEPAPTT sequence).

In other preferred embodiments where N is an integer greater than orequal to 3 (e.g., where N equals an integer from 3 through 200, or inmore preferred embodiments where N equals an integer from 5 through 50,or in even more preferred embodiments where N equals an integer from 10through 30), recombinant lubricin protein comprises the 22 amino acidsof SEQ ID NO: 28 joining the N-terminal-oriented 45 amino acids of SEQID NO: 26 to (N minus 2) repeat(s) of the 31 amino acids of SEQ ID NO:27, where the 10 amino acids of SEQ ID NO: 29 are C-terminal to the last31-amino-acid repeat of SEQ ID NO: 27.

TABLE 3 Sequence Frequencies in Preferred PRG4-LUB Proteins SEQ ID SEQID SEQ ID SEQ ID PRG4-LUB NO: 26 NO: 28 NO: 27 NO: 29 KEPAPTT ProteinN-end insert >--< >--< insert C-end repeats -LUB:1 1 0 0 0 4 -LUB:2 1 01 1 6 -LUB:3 1 1 1 1 9 -LUB:4 1 1 2 1 12 -LUB:5 1 1 3 1 15 -LUB:N 1 1 N− 2 1 3 × N

PRG4-LUB:N proteins in general have (3 times N) repeats of the KEPAPTTsequence in preferred embodiments where N equals the number ofrepetitive KEPAPTT-like sequences. Recombinant lubricin PRG4-LUB:5(having 3×N=3×5=15 copies of the KEPAPTT sequence in preferredembodiments) is the largest recombinant lubricin PRG4-LUB:N whosesequence is detailed herein. For recombinant lubricin of the presentinvention, however, the value N may be greater than 5, such as 7, 10,12, 15, 20, 25, 30, 40, 50, 100, 150, 200 or more.

In particular, proteins PRG4-LUB:1, PRG4-LUB:2, PRG4-LUB:3, PRG4-LUB:4,and PRG4-LUB:5 are detailed herein with 4, 6, 9, 12 and 15 perfectKEPAPTT sequences, respectively. However, it is possible to addincreasing numbers of KEPAPTT sequences by continuing the iterativeLub:N insert procedure described herein. We have provided detaileddescription for PRG4-LUB:N recombinant lubricins with relatively lownumbers of KEPAPTT or KEPAPTT-like sequences as compared with nativePRG4/lubricin protein because smaller proteins are easier to synthesizeand manipulate.

It may also be desirable to increase the number of KEPAPTT-likesequences over that seen in native PRG4 protein. This can beaccomplished either by continuing the iterative Lub:N insert proceduredescribed herein so that there are more than 78 KEPAPTT-like sequencesin the recombinant lubricin PRG4-LUB:N protein, or by beginning with anintact PRG4 cDNA, rather than an exon 6-deleted or an exon 6-diminishedversion of PRG4 cDNA. Thus any KEPAPTT-like sequences added will be inexcess of the number found in native PRG4 protein. Insert proceduresused for the generation of larger recombinant lubricin proteins from anintact PRG4 cDNA, as well as insert procedures that use an exon6-deleted or an exon 6-diminished version of PRG4 cDNA, are encompassedwithin the invention.

EXAMPLE 2 Expression and Purification of ‘Lub’ Protein

PRG4-Lub:1 cDNA construct (SEQ ID NO: 6; containing synthetic cDNAcassette-1 sequence) was expressed in a stably transfected, preadaptiveCHO DUKX cell line, purified from conditioned media, and solubilized inPBS containing 500 mM L-arginine hydrochloride as follows.

The PRG4-Lub:1 cDNA construct was expressed in a stably transfected CHODUKX cell line and the conditioned media was collected. A two litervolume of this conditioned media was filter concentrated undercompressed nitrogen gas (40 psi) using an AMICON® M2000™ filtration unitfitted with either a 10 kDa nominal molecular weight limit (NMWL), a 30kDa NMWL or a 100 kDa NMWL PALL FILTRON® OMEGA™ disc membrane. Media wasconcentrated to approximately a 100 ml volume, which was aspirated fromthe disc membrane. The disc membrane was then removed from the AMICON®M2000™ filtration unit. The “mucinous” retentate, which had accumulatedat the surface of the disc membrane, was harvested using a cell scraperand transferred to microcentrifuge tubes. The samples in themicrocentrifuge tubes were centrifuged at approximately 12,000×g for 10minutes, and the aqueous supernatant was removed. The remaining“lubricin-enriched” pellets were dissolved in phosphate buffered saline(PBS) containing 500 mM L-arginine hydrochloride. The L-argininehydrochloride concentration may range from 100 mM to 2.0 M.

Using the above procedure, PRG4-LUB:2 through PRG4-LUB:5 glycoproteins(and PRG4-LUB:N proteins where N=a nonnegative integer of 6 or more, aswell as other glycoproteins containing KEPAPTT-like sequences) areharvested directly from disc membranes, i.e., without purification ofthe concentrate remaining above disc membranes. That is, theserecombinant lubricin glycoproteins are isolated directly from discmembranes of 10 kDa NMWL, 30 kDa NMWL, or 100 kDa NMWL PALL FILTRON®OMEGA™ filtration units. In some instances, these glycoproteins may alsobe purified from the concentrate remaining above disc membranes throughchromatographic techniques or electrophoretic techniques or both.Recombinant lubricin proteins and glycoproteins may also be purifiedusing chromatography and other techniques known in the art (as, forexample, described in U.S. Pat. No. 6,433,142 for MSF proteins; seealso: Deutscher, 1990; and Scopes, 1994).

EXAMPLE 3 Immunohistochemistry

The cell source of lubricin in normal and osteoarthritic joints wasfurther investigated using immunohistochemical techniques. In addition,the presence of lubricin on other tissue surfaces, including pleura,pericardium, peritoneum, and meninges, was examined according to thefollowing methods.

Osteoarthritic cartilage and synovium were obtained by informed consentfrom patients undergoing knee replacement surgery. Other tissuesexamined were normal human synovium and normal non-human primate (NHP)synovium, cartilage, pleura, pericardium, peritoneum, meninges, brain,tendon, and ligaments, and canine normal and osteoarthritic meniscus,cartilage, synovium, ligament, and tendons. Tissues were fixed in 4%paraformaldehyde immediately after harvest or following 24 hoursincubation in media without and with supplemental monensin (5 μM). Forimmunohistochemical studies the tissues were fixed in 4%paraformaldehyde for 24 hours and 6-8 micron paraffin sections wereobtained. A subset of tissues were frozen in optical coherencetomography (OCT) freezing compound and cut at 5 to 10 micron intervalsfollowed by acetone fixation.

Immunohistochemical and immunofluorescent analyses utilized a purifiedpolyclonal rabbit anti-human lubricin antibody (Ab 06A10) generated byimmunization with a truncated form of recombinant lubricin andpurification on a protein A column. CD16 antibody (NEOMARKERS®, FremontCalif.) was used to identify macrophages (Fcy receptor III).CD106/VCAM-1 antibody (NEOMARKERS®) was used to label fibroblasts withincryostat sections. For control sections, an equivalent concentration ofRIgG (VECTOR LABS™, CA), MIgG₁ (DAKO®), and MIgG_(2a) (DAKO®) was usedconsecutively. The Dextran Technology System (ENVISION+™; DAKO®) wasused to visualize antibody binding and the sections were counterstainedwith Mayer's alum-hematoxylin. Immunofluorescence was performed usingthe above primary antibodies and probed with secondary antibodies (AlexaDyes—MOLECULAR PROBES™, Oregon) goat anti-rabbit Alexa dye at 546 nm andgoat anti-mouse Alexa dye at 488 nm. Fluorescent binding of the antibodywas detected with a NIKON® fluorescent microscope.

Lubricin was detected along the surfaces of normal and osteoarthritichuman articular cartilage and synovium. A thick layer of lubricincompletely coated the fibrillated osteoarthritic surface. CD106immunofluorescence showed strong cell membrane staining of the intimalfibroblasts of the synovium; lubricin protein was also visualized asstaining within synovial cells. Double immunostaining forCD106+lubricin, clearly showed co-localization within the intimalfibroblasts of the synovium. CD16 staining of synovial macrophagesdemonstrated the presence of these cells throughout the layers of thesynovium, but there was no co-localization with lubricin.

Staining of NHP and canine articular tissues (normal and OA) with thelubricin antibody showed lubricin coating the surface layer of thesynovium, cartilage, meniscus, and tendons. NHP cartilage also showedstrong immunoreactivity not only in the superficial zone cells but alsothe transitional zone cells without the addition of monensin to increaseintracellular stores of the glycoprotein. Cells lining the peritoneum,pericardium, and pleura also exhibited lubricin expression, though noimmunoreactivity was observed in the meninges or brain.

In summary, both normal and osteoarthritic synovium, tendon, meniscusand cartilage were coated by a substantial layer of lubricin. Theglycoprotein is clearly present on tissues within OA joints.Double-immunofluorescent staining of human OA synovium demonstrated thatthe intimal fibroblast synoviocytes were responsible for the synthesisof lubricin.

The localization of lubricin protein outside joint tissue has not beenpreviously described. A surface layer of lubricin was clearlydemonstrated on lung pleura, pericardium, and peritoneum. Lubricin isreputed to have a lubricating function within the synovial joint, butmay have multiple roles including, but not limited to, lubrication andanti-adhesive functions in other tissues. Supplementation of these othertissues with lubricin is a biotherapy encompassed within this invention.

EXAMPLE 4 Recombinant Lubricin as a Mechanical Lubricant

Recombinant lubricin could be used as a lubricant generally, e.g., withseals and bearings and the like. For example, U.S. Pat. No. 3,973,781entitled “Self-lubricating seal,” U.S. Pat. No. 4,491,331 entitled“Grooved mechanical face seal,” US4560174 entitled “Multi lip seal,” andU.S. Pat. No. 4,973,068 entitled “Differential surface roughness dynamicseals and bearings,” each describe seals of varying designs. Recombinantlubricin could be used as a lubricant with these seals.

In particular, recombinant lubricin could be used as a lubricant formedical devices, prostheses, and implants, particularly where abiocompatible lubricant is required. In addition, the applications neednot be medical, but could include applications in environmentallysensitive contexts where a biocompatible lubricant may be desirable.

EXAMPLE 5 Recombinant Lubricin Compositions

A recombinant lubricin of the present invention may be used in apharmaceutical composition when combined with a pharmaceuticallyacceptable carrier. Such a composition may also contain (in addition toprotein and a carrier) diluents, fillers, salts, buffers, stabilizers,solubilizers, and other materials well known in the art. The term“pharmaceutically acceptable” means a non-toxic material that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s). The characteristics of the carrier will depend onthe route of administration. The pharmaceutical composition of theinvention may also contain cytokines, lymphokines, or otherhematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, anderythropoietin. The pharmaceutical composition may further contain otheragents which either enhance the activity of the protein or complementits activity or use in treatment. Such additional factors and/or agentsmay be included in the pharmaceutical composition to produce asynergistic effect with protein of the invention, or to minimize sideeffects. Conversely, protein of the present invention may be included informulations of the particular cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent to minimize side effects.

Use of recombinant lubricin protein for intra-articular supplementationin combination with the previously described polymeric hyaluronan (HA)and higher molecular weight hylans is particularly preferred. Otherpreferred combinations for use in intra-articular supplementationinclude the use of recombinant lubricin protein with anesthetics (e.g.,lidocaine), steroids (e.g., triamcinolone hexacetonide), orradioisotopes (e.g., yttrium). Other preferred combinations for use inintra-articular supplementation may include autologous or heterologouscell preparations (e.g., of cultured chondrocytes, synoviocytes, or stemcells, whether autologously or heterologously derived).

A recombinant lubricin of the present invention may be active inmultimers (e.g., heterodimers or homodimers) or complexes with itself orother proteins. As a result, pharmaceutical compositions of theinvention may comprise a protein of the invention in such multimeric orcomplexed form.

A pharmaceutical composition of the invention may be in the form of acomplex of the recombinant lubricin protein(s) of present inventionalong with protein or peptide antigens. The protein and/or peptideantigen will deliver a stimulatory signal to both B and T lymphocytes. Blymphocytes will respond to antigen through their surface immunoglobulinreceptor. T lymphocytes will respond to antigen through the T cellreceptor (TCR) following presentation of the antigen by MHC proteins.MHC and structurally related proteins including those encoded by class Iand class II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunolgobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.

A pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. No. 4,235,871, U.S. Pat. No.4,501,728, U.S. Pat. No. 4,837,028, and U.S. Pat. No. 4,737,323.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a subject (e.g., a mammal) having a condition to betreated. Protein of the present invention may be administered inaccordance with the method of the invention either alone or incombination with other therapies such as treatments employing cytokines,lymphokines, other hematopoietic factors, or cell-based supplements.When co-administered with one or more cytokines, lymphokines, otherhematopoietic factors, or cell-based supplements, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or cell-based supplement, or sequentially.If administered sequentially, the attending physician will decide on theappropriate sequence of administering protein of the present inventionin combination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors, or cell-basedsupplement.

Administration of protein of the present invention used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such ascutaneous, subcutaneous, intraperitoneal, parenteral or intravenousinjection, or, in some instances, oral ingestion, inhalation, topicalapplication. Administration to a patient by injection into joint tissueis generally preferred (Schumacher, 2003).

When a therapeutically effective amount of protein of the presentinvention is administered orally, protein of the present invention willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%protein of the present invention, and preferably from about 25 to 90%protein of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein of the present invention, and preferably from about 1to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forexample, injection in association with, or in combination with,lidocaine or other local anesthetic, steroids or adrenocorticoids, HAand/or hylans, or radioisotopes are all encompassed within by thepresent invention.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight depending on the method ofadministration and the exact therapeutic course implemented.

If administered intravenously, the duration of intravenous therapy usinga pharmaceutical composition comprising recombinant lubricin of thepresent invention will vary, depending on the severity of the diseasebeing treated and the condition and potential idiosyncratic response ofeach individual patient. It is contemplated that the duration of eachapplication of the protein of the present invention may be in the rangeof 12 to 24 hours of continuous intravenous administration. Ultimatelythe attending physician will decide on the appropriate duration ofintravenous therapy using the pharmaceutical composition of the presentinvention.

For compositions of the present invention which are useful for bone,cartilage, tendon or ligament therapy, the therapeutic method includesadministering the composition topically, systematically, or locally asan implant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to the site of bone,cartilage or tissue damage. Topical administration may be suitable forin some wound healing and tissue repair contexts. Therapeutically usefulagents which may also optionally be included in the composition asdescribed above, may alternatively or additionally, be administeredsimultaneously or sequentially with the composition comprisingrecombinant lubricin protein of the invention in the methods of theinvention. Preferably the composition would include a matrix capable ofdelivering the protein-containing composition to the site of bone and/orcartilage damage, possibly capable of providing a structure for thedeveloping bone and cartilage, and optimally capable of being resorbedinto the body. Such matrices may be formed of materials presently in usefor other implanted medical applications.

If a matrix is used, the choice of matrix material is based onbiocompatibility, biodegradability, mechanical properties, cosmeticappearance and interface properties. The particular application of thecompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sintered hydroxapatite,bioglass, aluminates, or other ceramics. Matrices may be comprised ofcombinations of any of the above mentioned types of material, such aspolylactic acid and hydroxyapatite or collagen and tricalciumphosphate.The bioceramics may be altered in composition, such as incalcium-aluminate-phosphate and processing to alter pore size, particlesize, particle shape, and biodegradability.

In further compositions, proteins of the invention may be combined withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in question. These agents include variousgrowth factors such as epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals such as cats and dogs,laboratory animals such as mice and rats, as well as horses, in additionto humans, are particularly desired subjects or patients for suchtreatment with recombinant lubricin proteins of the present invention.

The dosage regimen of a protein-containing pharmaceutical composition tobe used in tissue regeneration will be determined by the attendingphysician considering various factors which modify the action of theproteins, e.g., amount of tissue weight desired to be formed, the siteof damage, the condition of the damaged tissue, the size of a wound,type of damaged tissue (e.g., cartilage or tendon), the patient's age,sex, and diet, the severity of any infection, time of administration andother clinical factors. The dosage may vary with the type of matrix usedin the reconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a subject (e.g., a mammal).Polynucleotides of the invention may also be administered by other knownmethods for introduction of nucleic acid into a cell or organism(including, without limitation, in the form of viral vectors or nakedDNA).

Cells may also be cultured ex vivo in the presence of nucleic acids orproteins of the present invention in order to proliferate or to producea desired effect on or activity in such cells. Treated cells can then beintroduced in vivo for therapeutic purposes.

EXAMPLE 6 Anti-Lubricin Antibodies

Recombinant lubricin protein of the invention may also be used toimmunize animals to obtain polyclonal and monoclonal antibodies whichspecifically react with the protein or, in some embodiments, its nativecounterparts. Such antibodies may be obtained using either completerecombinant lubricin protein or fragments thereof as an immunogen. Thepeptide immunogens additionally may contain a cysteine residue at thecarboxyl terminus, and are conjugated to a hapten such as keyhole limpethemocyanin (KLH). Methods for synthesizing such peptides are known inthe art (for example, as in Merrifield, 1963; and Krstenansky et al.,1987). Monoclonal antibodies binding to recombinant lubricin protein ofthe invention may be useful diagnostic agents for the immunodetection ofrelated proteins. Neutralizing monoclonal antibodies binding to theserelated proteins may also be useful therapeutics for both conditionsassociated with lubricin or, in some cases, in the treatment of someforms of cancer where abnormal expression of lubricin may be involved(e.g., in synoviomas).

In addition to antibodies which are directed to the polypeptide core ofa recombinant lubricin protein, an antibody directed to a sugar portionor to a glycoprotein complex of recombinant lubricin protein isdesirable. In order to generate antibodies which bind to glycosylatedrecombinant lubricin (but not to a deglycosylated form), the immunogenis preferably a glycopeptide, the amino acid sequence of which spans ahighly glycosylated portion of the recombinant lubricin, e.g., arepetitive KEPAPTT-like sequence. Shorter glycopeptides, e.g., 8-15amino acids in length, within the same highly glycosylated region, arealso used as immunogens. Methods of generating antibodies to highlyglycosylated biomolecules are known in the art (for example, asdescribed by Schneerson et al., 1980).

EXAMPLE 7 Recombinant Lubricin Delivery

Standard methods for delivery of recombinant lubricin are used. Forintra-articular administration, recombinant lubricin is delivered to thesynovial cavity at a concentration in the range of 20-500 μg/ml in avolume of approximately 0.1-2 ml per injection. For example, 1 ml of arecombinant lubricin at a concentration of 200-300 μg/ml is injectedinto a knee joint using a fine (e.g., 14-30 gauge, preferably 18-26gauge) needle. The compositions of the invention are also useful forparenteral administration, such as intravenous, subcutaneous,intramuscular, or intraperitoneal administration, and, in preferredembodiments, onto the surfaces of the peritoneal, pericardium, orpleura.

Proper needle placement is critical for the efficacy of recombinantlubricin protein that is delivered by injection in joint therapies(Schumacher, 2003). Proper needle placement may be facilitated throughthe use of ultrasound technology. Successful injections are more commonafter successful aspiration of fluid is obtained. A supralateralapproach into the suprapatellar pouch has been suggested to provide themost reliable access to knee joint space. In addition to administeringrecombinant lubricin by intra-articular injection, nucleic acidsencoding recombinant lubricin (e.g., in gene therapy applications) maybe administered to a synovial cavity by intra-articular injection.

For prevention of surgical adhesions, recombinant lubricins describedherein are administered in the form of gel, foam, fiber or fabric. Arecombinant lubricin formulated in such a manner is placed over andbetween damaged or exposed tissue interfaces in order to preventadhesion formation between apposing surfaces. To be effective, the gelor film must remain in place and prevent tissue contact for a longenough time so that when the gel finally disperses and the tissues docome into contact, they will no longer have a tendency to adhere.Recombinant lubricin formulated for inhibition or prevention of adhesionformation (e.g., in the form of a membrane, fabric, foam, or gel) areevaluated for prevention of post-surgical adhesions in a rat cecalabrasion model (Goldberg et al., 1993). Compositions are placed aroundsurgically abraded rat ceca, and compared to non-treated controls(animals whose ceca were abraded but did not receive any treatment). Areduction in the amount of adhesion formation in the rat model in thepresence of recombinant lubricin formulation compared to the amount inthe absence of the formulation indicates that the formulation isclinically effective to reduce tissue adhesion formation. In contextswhere tissue adhesion is desired (e.g., where healing of cartilagefissures is desired), however, use of recombinant lubricin may be bestavoided. Providing lubrication to cartilage surfaces impairscartilage-cartilage integration (Schaefer et al., 2004).

Recombinant lubricins are also used to coat artificial limbs and jointsprior to implantation into a mammal. For example, such devices may bedipped or bathed in a solution of a recombinant lubricin, e.g.,following methods described in U.S. Pat. No. 5,709,020 or U.S. Pat. No.5,702,456. Care should be exercised, however, in the in vivo use ofrecombinant lubricin in providing lubrication near a prostheses. Amarked upregulation in PRG4 gene expression (i.e., MSF gene expression)has been reported to be associated with prosthesis loosening; lubricincould disturb the tight interaction between bone and prosthesis andthereby contribute to prosthesis loosening (Morawietz et al., 2003).

EXAMPLE 8 OA Model

In order to assess the efficacy of intra-articular administration oflubricin preparations, a murine model of osteoarthritis/cartilageerosion is prepared. For surgical induction of osteoarthritis, mice areanesthetized with 250 mg/kg intraperitoneal tribromoethanol (SIGMA®Chemical), and knees are prepared for aseptic surgery. A longitudinalincision medial to the patellar ligament is made, the joint capsule isopened, and the meniscotibial ligament (anchoring the medial meniscus tothe tibial plateau) is identified. In a subset of animals, no furthermanipulation is performed, and this group is considered sham operated.In the experimental group the medial meniscotibial ligament istransected resulting in destabilization of the medial meniscus (DMM). Inboth sham and DMM animals, the joint capsule and subcutaneous layer aresutured closed separately and the skin is closed by application ofNEXABAND® S/C tissue adhesive (Abbott, North Chicago, Ill.).Buprenorphine (BUPRENEX®; Reckitt & Coleman, Kingston-upon-Hull, UK) isadministered pre- and post-operatively,

Recombinant lubricin preparations are administered by intra-articularinjection using a 30 gauge needle. Injections of 5-10 microliters perknee joint are administered one week post surgery. Additional injectionsare optionally administered on a weekly basis. Animals are sacrificed bycarbon dioxide at 4 weeks post-operatively and at 8 weekspost-operatively.

In order to assess the progression and severity of osteoarthritis,intact knee joints are placed into 4% paraformaldehyde for 24 hours,then decalcified in EDTA/polyvinylpyrrolidone for five days. Joints areembedded in paraffin and 6-μm frontal sections obtained through theentire joint. Slides are stained with Safranin O-fast green and gradedat 70-μm intervals through the joint using a modification of asemi-quantitative scoring system (Chambers et al., 2001) in which“0”=normal cartilage; “0.5”=loss of Safranin O without structuralchanges; “1”=roughened articular surface and small fibrillations;“2”=fibrillation down to the layer immediately below the superficiallayer and some loss of surface lamina; “3”=mild (<20%); “5”=moderate(20-80%); and “6”=severe (>80%) loss of non-calcified cartilage. Scoresof “4” (erosion to bone) are not a feature of this model. All quadrantsof the joint (medial tibial plateau, medial femoral condyle, lateraltibial plateau, and lateral femoral condyle) are scored separately. Aminimum of 12 levels are scored by blinded observers for each kneejoint. Scores are expressed as the maximum histologic score found ineach joint or the summed histologic scores. The summed score representsthe additive scores for each quadrant of the joint on each histologicsection through the joint. This method of analysis enables assessment ofseverity of lesions as well as the surface area of cartilage affectedwith OA-like lesions (Glasson et al., 2004).

REFERENCES

(1) Chambers et al., 2001, Arthritis Rheum. 44: 1455-65; (2) Deutscher,1990, Methods in Enzymology, Vol. 182: Guide to Protein Purification,Academic Press; (3) Espallargues and Pons, 2003, Int'l J. Tech. Assess,Health Care 19: 41-56; (4) Flannery et al., 1999, Biochem. Biophys. Res.Comm. 254: 535-41; (5) Glasson et al., 2004, Arthritis Rheum. 50:2547-58; (6) Goldberg et al., 1993, In: Gynecologic Surgery and AdhesionPrevention, Willey-Liss, pp. 191-204; (7) Hills, 2002, J. Rheumatology29: 200-01; (8) Ikegawa et al., 2000, Cytogenet. Cell Genet. 90:291-297; (9) Jay et al., 2001, J. Orthopaedic Research 19: 677-87; (10)Jay et al., 2002, Glycoconjugate Journal 18: 807-15; (11) Krstenansky etal., 1987, FEBS Lett. 211: 10-16; (12) Marcelino et al., 1999, NatureGenetics 23: 319-322; (13) Merberg et al., 1993, Biology of Vitronectinsand their Receptors, Pressner et al. (eds.): Elsevier SciencePublishers, pp. 45-53; (14) Merrifield, 1963, J. Amer. Chem. Soc. 85:2149-54; (15) Morawietz et al., 2003, Virchows Arch. 443: 57-66; (16)Rees et al., 2002, Matrix Biology 21: 593-602; (17) Schneerson et al.,1980, J. Exp. Med. 152: 361-76; (18) Scopes, 1994, Protein Purification:Principles and Practice (3^(rd) edition), Springer Verlag; (19) Schaeferet al., 2004, Biorheology 41: 503-508; (20) Schumacher, 2003, Arthritis& Rheumatism 49: 413-20; (21) Tatusova and Madden, 1999, FEMS MicrobiolLett. 174: 247-50; (22) Wobig et al., 1998, Clin. Ther. 20: 410-23; and(23) Wobig et al., 1999, Clin. Ther. 21: 1549-62.

1.-10. (canceled)
 11. An isolated protein comprising the amino acidsequence of SEQ ID NO:
 15. 12-32. (canceled)
 33. The protein of claim11, wherein the protein is O-linked to β-(1-3)-Gal-GalNac.
 34. Acomposition comprising a therapeutically effective amount of a proteinof claim 33 in a pharmaceutically acceptable carrier.
 35. Thecomposition of claim 34, additionally comprising hyaluronan or hylan.36. The composition of claim 34, wherein the composition is formulatedfor injection.
 37. The composition of claim 34, wherein the compositionis encapsulated.
 38. The composition of claim 37, wherein thecomposition is encapsulated in an implantable drug delivery matrix. 39.The composition of claim 34, wherein the composition further comprises alocal anesthetic.
 40. The composition of claim 34, wherein thecomposition further comprises a cytokine.
 41. The composition of claim34, wherein the composition further comprises a lymphokine.
 42. Thecomposition of claim 34, wherein the composition further comprises athrombolytic factor.
 43. The composition of claim 34, wherein thecomposition further comprises an antithrombolytic factor.
 44. Thecomposition of claim 34, wherein the composition further comprises ananti-inflammatory agent.
 45. The composition of claim 34, wherein thecomposition further comprises an antibiotic.
 46. The composition ofclaim 34, wherein the composition further comprises a growth factor.